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Comparison between different trypsin digestion procedures used for LC-ESI-MS peptide mapping
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(English)Manuscript (Other (popular science, discussion, etc.))
Identifiers
URN: urn:nbn:se:uu:diva-94669OAI: oai:DiVA.org:uu-94669DiVA: diva2:168610
Available from: 2006-08-30 Created: 2006-08-30 Last updated: 2011-03-21
In thesis
1. Liquid Chromatography Coupled to Mass Spectrometry: Implementation of Chemometric Optimization and Selected Applications
Open this publication in new window or tab >>Liquid Chromatography Coupled to Mass Spectrometry: Implementation of Chemometric Optimization and Selected Applications
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Liquid chromatography (LC) coupled to mass spectrometry (MS) offers highly selective and sensitive analysis of a wide variety of compounds. However, the use of hyphenated experimental set-ups implies that many parameters may have an effect on the studied response. Therefore, in order to determine optimized experimental conditions it is of vital importance to incorporate systematic procedures during method development. In this thesis, a generic stepwise optimization strategy is proposed that aims at high chromatographic quality, as well as high mass spectrometric response. The procedure comprises (i) screening experiments to identify the most important parameters, (ii) LC studies to ensure sufficient chromatographic separation, (iii) extended infusion experiments in order to maximize precursor signal(s), and in the case of tandem MS (iv) extended infusion experiments to determine optimal conditions for collision induced dissociation and when applicable also ion trap settings. Experimental design and response surface methodology is used throughout the procedure.

Further, the general applicability of LC-MS is demonstrated in this thesis. Specifically, a novel quantitative column-switched LC-MS method for ferrichrome, ferrichrysin and ferricrocin determination is presented. Using the method it was shown how the siderophore content varies with depth in podzolic soil profiles in the north and south of Sweden. The parallel approach using LC coupled to both inductively coupled plasma (ICP) mass spectrometry, and electrospray ionization (ESI) tandem MS is also evaluated as a tool to identify unknown siderophores in a sample. Additionally, different trypsin digestion schemes used for LC-ESI-MS peptide mapping were compared. By multivariate data analysis, it was clearly shown that the procedures tested induce differences that are detectable using LC-ESI-MS. Finally, the glutathione S-transferase catalyzed bioactivation of the prodrug azathioprine was verified using LC-MS.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2006. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 201
Keyword
Analytical chemistry, liquid chromatography, mass spectrometry, tandem mass spectrometry, electrospray ionization, inductively coupled plasma, chemometrics, optimization, multivariate data analysis, design of experiments, response surface model, siderophore, azathioprine, peptide, Analytisk kemi
Identifiers
urn:nbn:se:uu:diva-7071 (URN)91-554-6612-5 (ISBN)
Public defence
2006-09-28, Room B:21, BMC, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2006-08-30 Created: 2006-08-30 Last updated: 2009-03-24Bibliographically approved

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